The increases of life expectancy seen throughout the developed world are a valuable indicator for the state of modern medicine. However, a less quantifiable measure is quality of life during those additional years. The incidence of skeletal injuries along with age related conditions such as osteoporosis and osteoarthritis provide one such metric. Treatments which involve increasing bone density or fracture healing are areas where the regenerative potential of mesenchymal stem cells (MSCs) could prove beneficial [1, 2]. The controlled osteogenesis of MSCs through mechanical means has already been demonstrated through several methods including passive and active means. Passive methods such as altered substrate topography and stiffness provide one mechanism based on altering the adhesion profile [3-7], whilst active methods include exposure to variations of force from external sources [8-13]. Centrifuge, vibration and shear flow have all provided increases in osteogenesis through external modulation of the force experienced by the cell structure. Work by D Ingber has posed a potential description for the highly tuned mechano-sensitive nature of the cytoskeleton when considered as one sensory unit [14, 15]. The varying mechanisms which induce osteogenesis are still being explored and it is now accepted that stimulating cells on the macro and micro scale, using extracellular scaffolds and biomimetic polymers to induce differentiation, is of significant use [16-19]. However, there is now a growing understanding that stimulation and moreover manipulation of the cells at the nano-scale can have a complementary and even greater downstream effect on stem cell differentiation and control [20, 21].
One of the key components which these studies have highlighted is the importance of focal adhesions and integrins as transducers in the osteogenic mechano-transductive process [22, 23]. Altering topography can allow larger more fibrillary focal adhesions to form [24, 25], and the resulting process which drives the osteogenesis of MSCs can include focal adhesion kinase (FAK) signaling. This biological sensory cascade can subsequently be augmented through the mitogen activated protein kinase (MAPK) pathway (including extracellular signal regulated kinase (ERK), and activate Runt-related transcription factor 2 (RUNX2) and osterix. These transcription factors are linked with osteogenic differentiation, along with the genes osteocalcin (OCN), osteonectin (ONN), alkaline phosphatase (ALP) [26-28].
The use of vibration as a mechano-transductive stimulus has been explored with varied vibrational parameters [29-31]. Vibration of periodontal ligament stem cells at 50 Hz (sine wave) with peak acceleration of 3 g showed increased markers of osteogenesis [32] whilst another study of adipose stem cells stimulated using 50 and 100 Hz square waves with accelerations of 3 g showed increased levels of ALP activity and mineral deposition, however not at the same level produced by osteogenic media [33]. Although interesting, neither study mentions the vibration amplitude used. If the focal adhesion complex is important to producing osteogenesis then nanometer sized amplitudes, aimed at the length scale of the integrin complex could find increased stimulation of focal adhesion related signaling.
The use of piezoelectric actuators to produce vibrations of nanometer amplitude has now become a focus for research [21, 34, 35]. Curtis et al. and Nikukar et al. showed that endothelial cells and MSCs are sensitive to amplitudes of tens of nanometers. The use of single actuator, single petri dish devices showed the ability to produce accurate vertical vibration over the entire growth surface. The result of these studies have shown that nanovibration of endothelial cells at low frequencies (1-10 Hz) indicates that protein expression and gene expression linked with an increasing endothelial phenotype are up regulated. In addition experiments incorporating white noise or frequency sweeps show the importance of a coherent signal in producing the biological response. The stimulation of MSCs at higher frequencies (500-1000 Hz), also utilizing nano vibration, have shown the osteogenesis of MSCs. An expansion of this study by Pemberton, Childs et al. up to 5000 Hz used in tandem with nano-topography shows that the two stimuli are not additive with vibration alone producing greater up-regulation of osteogenesis. A comparison of 1, 3 and 5000 Hz also shows that little extra up-regulation of the previous markers is shown above 1000 Hz [36].